Fanconi Anemia Cells Research Articles

446. Stable Correction of Alkylator Resistance in Fanconi Anemia Cells By a Mitotically Replicating Non-Integrating Lentiviral Vector

Fanconi anemia (FA) is an inherited multisystem disorder leading to symptomatic hematopoietic defects by early school age in most patients. Observations of lymphoid mosaicism and spontaneous intragenic correction of stem cells leading to clonal hematopoiesis in a small number of patients indicate that phenotypically corrected cells enjoy a survival advantage over FA deficient cells. Accordingly, FA is a leading genetic disorder target for HSPC directed gene therapy. However, reports over the insertional complications of commonly used integrating vectors continue to raise concerns, while available non-integrating lentivectors (NILVs) are generally lost from dividing hematopoietic stem cells. We recently showed that the incorporation of human Scaffold/Matrix Attachment Region (S/MAR) sequences in NILVs results in the cellular retention of NILV episomes in actively dividing cell lines and HSPCs without selection. Southern analysis, Alu PCR and episome specific PCR all confirmed long-term episomal existence of this vector without genomic integration (NAR. 2014; PMID: 24474068). Here, we designed and tested an episome lentivector to phenotypically correct FA cells (aNILV GFP-FANCC). Results showed the vector expressing the human FANCC ORF fused with GFP could be readily packaged, with stable persistence of vector episomes and sustained GFP expression. Taking advantage of the selective expansion of phenotypically corrected cells, we transduced a FANCC -deficient human cell line pD331 by using aNILV-GFP-FANCC vector. Exaggerated sensitivity to DNA alkylating agents (e.g. Mitomycin C, MMC) is a hallmark of FA deficient cells and can be used as a selection agent. At week 2, we observed 15% retention of GFP expressing cells in MMC treated pD331-aNILV-FANCC population versus 3% retention in non-treated pD331-aNILV-FANCC cells. Long-term maintenance of cells up to 6 weeks under MMC selection resulted the enrichment of corrected cells up to 95% that is comparable to the enrichment observed by integrating LV expressingGFP-FANCC in pD331 cells under MMC selection. Molecular studies of aNILV episomes from long-term cultures by episome specific PCR revealed LTR episome persistence. Integration analysis by Alu-PCR from the bulk population with Southern analysis of clones is ongoing. We already reported that episomal S/MAR NILV transduced short-term murine hematopoietic stem and progenitor cells and are currently characterizing this vector in a murine model of Fanconi Anemia. In conclusion, epsiome maintenance under selective pressure and reversal of canonical alkylator sensitivity in FA cells make this novel aNILV-GFP-FANCC vector a consideration for expanded preclinical exploration.

A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea-induced DNA damage.

Fanconi anemia (FA) is a genetic disease of bone marrow failure, cancer susceptibility, and sensitivity to DNA crosslinking agents. FANCD2, the central protein of the FA pathway, is monoubiquitinated upon DNA damage, such as crosslinkers and replication blockers such as hydroxyurea (HU). Even though FA cells demonstrate unequivocal sensitivity to crosslinkers, such as mitomycin C (MMC), we find that they are largely resistant to HU, except for cells absent for expression of FANCD2. FANCD2, RAD51 and RAD18 form a complex, which is enhanced upon HU exposure. Surprisingly, although FANCD2 is required for this enhanced interaction, its monoubiquitination is not. Similarly, non-ubiquitinated FANCD2 can still support proliferation cell nuclear antigen (PCNA) monoubiquitination. RAD51, but not BRCA2, is also required for PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion (TLS) polymerase PolH chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knockdown cells and RAD51 siRNA knockdown cells, and PolH knockdown results in HU sensitivity only. Our data suggest that FANCD2 and RAD51 have an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with MMC treatment, suggesting a non-canonical function for the FA pathway in response to different types of DNA damage.

Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers.

Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual.

Impaired immune response to Candida albicans in cells from Fanconi anemia patients

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and cancer predisposition. Several studies show alterations of the immunological status of FA patients including defects in peripheral blood lymphocyte subsets, serum immunoglobulin levels, and inflammatory cytokines. However scanty information is available on the response of FA cells to specific infectious antigens. In this work we examined the response of FA cells to different immunological stimuli and found a defective response of IL-1β, TNF-α and IL-17 to Candida albicans stimulation thus pointing to a potentially impaired response to fungal infections of FA patients.

Fanconi anemia (FA) and crosslinker sensitivity: Re-appraising the origins of FA definition.

The commonly accepted definition of Fanconi anemia (FA) relying on DNA repair deficiency is submitted to a critical review starting from the early reports pointing to mitomycin C bioactivation and to the toxicity mechanisms of diepoxybutane and a group of nitrogen mustards causing DNA crosslinks in FA cells. A critical analysis of the literature prompts revisiting the FA phenotype and crosslinker sensitivity in terms of an oxidative stress (OS) background, redox-related anomalies of FA (FANC) proteins, and mitochondrial dysfunction. This re-appraisal of FA basic defect might lead to innovative approaches both in elucidating FA phenotypes and in clinical management.

ROMO1 regulates RedOx states and serves as an inducer of NF-κB-driven EMT factors in Fanconi anemia

Fanconi anemia (FA) is a rare genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. Besides the well-established role of FA genes in DNA damage and repair pathways, recent reports have revealed an overproduction of epithelial to mesenchymal transition (EMT) factors via a NF-κB-dependent mechanism that results in the proliferation of neighboring tumor cells and FA cells have also been shown to possess damaged mitochondria, accompanied by altered RedOx pathways. This study has focused on reactive oxygen species Modulator-1 (ROMO1), an oncomarker and mitochondrial membrane protein, which is known to be associated with cancer growth and in the modulation of RedOx states in some cancer models. Here, we reveal the role of ROMO1 and demonstrate its link in regulating RedOx states and in the activation of NF-κB-dependent EMT factors in FA.

Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells.

Fanconi's anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin-C (MMC) on FA patient-derived lymphoblastoid cell lines from groups FA-A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC-induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well-known role in replication fork stalling during S-phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457-467, 2015. © 2015 Wiley Periodicals, Inc.

Dysregulated Ca2+ homeostasis in Fanconi anemia cells.

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca2+]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca2+ mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca2+]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca2+ stores. The defects associated with the altered Ca2+ homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

Bone Marrow Failure in Fanconi Anemia from Hyperactive TGF-β Signaling

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life due to attrition of hematopoietic stem and progenitor cells (HSPCs). FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutants in one of sixteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA may result, directly or indirectly, from hyperactivation of cell autonomous or microenvironmental growth suppressive pathways induced due to genotoxic stress. Recent studies suggest that one suppressive pathway may be the hyperactive p53 response observed in HSPCs from FA patients.In order to further identify suppressive mechanisms accounting for bone marrow failure in FA, we performed a whole genome-wide shRNA screen in FA cells. Specifically, we screened for candidate genes whose knockdown would rescue cellular growth inhibition and genotoxic stress induced by a DNA cross-linking agent mitomycin C (MMC). We transduced a FA-deficient human fibroblast line with pools of shRNAs and screened for rescue of MMC-inhibited growth. Selected shRNA inserts were identified by next generation sequencing.The top hits in the screen were shRNAs directed against multiple components of the TGF-β signaling pathway. Consistent with this, disruption of the TGF-β signaling pathway by shRNA/sgRNA-mediated knockdown of SMAD3 or TGFR1 (downstream components of the TGF- β pathway) rescued growth of multiple cell lines from several FA complementation groups in presence of genotoxic agents (e.g. MMC or acetaldehyde). Pharmacologic inhibition of the TGF- β pathway using small molecule inhibitors resulted in improved survival of FA-deficient lymphoblast cells in presence of MMC or acetaldehyde, suggesting that a hyperactive, TGF-β-mediated, suppression pathway may account, at least in part, for reduced FA cell growth. Interestingly, genes encoding TGF-β pathway signaling components were highly expressed in the bone marrow from FA patients and FA mice. Moreover, disruption of the TGF- β pathway by shRNA-mediated knockdown of SMAD3 rescued the growth defects of primary HSPCs from FA-deficient murine bone marrow. To further implicate the TGF-β pathway, we established primary stromal cell lines from the bone marrow of FA-deficient mice as well as human FA patients. We confirmed that TGF-β signaling was hyperactive in these stroma cells resulting in growth suppression and elevated phospho-ERK levels due to non-canonical signaling of the pathway. Inhibitors of TGF-β signaling partially rescued the growth defects and reduced phospho-ERK levels in these FA stroma cells.The deficiency of FA DNA repair pathway leads to cellular defects in homologous recombination (HR) repair and hyperactivation of toxic non-homologous end joining (NHEJ)-mediated repair. We therefore tested whether inhibition of the TGF-β pathway in FA cells could rescue HR defects and account for the improvement of FA cellular growth. Interestingly, disruption of the TGF-β signaling pathway caused a decrease in NHEJ activity. Disruption of the TGF-β pathway also resulted in reduced MMC-mediated DNA damage and increased HR.Taken together, our results demonstrate that primary FA hematopoietic and bone marrow stromal cells exhibit hyperactive TGF-β signaling accounting at least in part for the bone marrow failure in FA. Inhibitors of the TGF-β signaling pathway may therefore be useful in the clinical treatment of patients with bone marrow failure and Fanconi anemia. DisclosuresNo relevant conflicts of interest to declare.

Differential Response of Fanconi Anemia Hematopoietic Stem and Progenitor Cells to Oxidative and Oncogenic Stresses

Members of the Fanconi anemia (FA) protein family are involved in DNA damage response. A common damage to DNA in vivo is oxidative stress, and compelling evidence suggests that FA cells are in an in vivo pro-oxidant state. In response to oncogenic activation, normal cells induce genetically encoding programs that prevent deregulated proliferation and thus protect multicellular organisms from cancer progression. How FA cells respond to oxidative DNA damage and oncogenic stress is largely unknown. By employing an in vivo stress-response model expressing the Gadd45b-luciferase transgene, we show here that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc differentially responded to oxidative and oncogenic stresses. Compared to wild-type controls, Fanca-/- or Fancc-/- HSPCs exhibited a persistent response to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. In contrast, using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we identify a short-lived response of FA HSPCs to oncogenic insults both in vitro and in vivo. Mechanistic studies revealed that loss of Fanca or Fancc impaired oncogenic stress-induced senescence (OIS), and genetic correction of Fanca or Fancc deficiency restored OIS in HSPCs. Furthermore, FA deficiency compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Finally, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca-/- mice prolonged oncogenic response and delayed leukemia development in recipient mice. Taken together, our study demonstrates differential responses of HSPCs to oxidative and oncogenic stresses and identifies the FA pathway as an integral part of this versatile cellular mechanism. DisclosuresNo relevant conflicts of interest to declare.

Inducible Loss of the Fanconi Anemia Pathway in iPSC Causes Rapid Cell Cycle Arrest and Apoptosis through ATM/ATR and p53 Signaling

As the source of all cells in the developing embryo proper, embryonic stem cells (ESC) bear the unique responsibility to prevent mutations from being propagated throughout the entire organism and the germ line. It is likely for this reason that ESC and induced pluripotent stem cells (iPSC) maintain a dramatically lower mutation frequency than cultured somatic cells. Multiple mechanisms for this enhanced genomic surveillance have been proposed, including hypersensitivity of DNA damage response signaling pathways and increased activity of error-free DNA repair pathways, such as homologous recombination. However, the effect of loss of function of DNA repair pathways in these cells remains poorly understood. The Fanconi Anemia (FA) pathway is a DNA repair pathway that is required for the repair of DNA interstrand crosslink damage and also promotes repair of DNA double-strand breaks by homologous recombination . Genetic defects in this pathway cause a disease characterized by bone marrow failure and extreme cancer incidence. Several recent studies have revealed that the FA pathway is required for efficient somatic cell reprogramming to iPSC and suggest that FA cells undergo cell death during this process. Another recent study found that the growth of FA patient-specific iPSC was attenuated with a G2/M arrest when compared to control iPSC, suggesting that these cells arrest upon failed DNA repair. In this study, we sought to determine the effects of acute loss of function of the FA pathway in iPSC through the generation of FA patient-derived iPSC with inducible complementation of the defective FA gene.Fibroblasts were cultured from skin biopsies of multiple FA patients and transduced with a lentiviral vector expressing the complementing FA gene product under DOX-inducible control. Cells were then reprogrammed to iPSC using episomal transfection. These cells formed iPSC colonies only when reprogramming was carried out in the presence of DOX, confirming that the FA pathway is required for efficient reprogramming. Once cell lines were obtained, DOX-dependent FA functionality was verified based on FANCD2 monoubiquitination and nuclear focus formation after treatment with DNA damaging agents. We then cultured the iPSC for extended periods of time in the presence and absence of DOX. Interestingly, the cultures underwent profound cell death and cell cycle arrest within 7 days of DOX-withdrawal and completely failed to expand after one passage. EdU cell cycle analysis confirmed cell cycle arrest in the G2/M phase. Furthermore, cleaved caspase 3 staining confirmed that the number of apoptotic cells increased by 3-fold in the -DOX culture. Despite these effects, cells cultured in both the presence and absence of DOX formed teratomas in nude mice, thus indicating the maintenance of full differentiation capacity in the absence of the FA pathway. In order to determine the mechanisms underlying G2/M arrest and cell death, expression of p53 and its target genes was detected by both western blot analysis and qRT-PCR. Only a slight increase in p53 activation was observed by 7 days post DOX-withdrawal. Furthermore, knockdown of p53 resulted in rescue from apoptosis to normal levels but not rescue from cell cycle arrest. Increased ATM and ATR DNA damage sensor kinase activities were also detected in –DOX cells, concominant with increased phosphorylation of the ATM-target Chk2 and reduced abundance of the G2/M checkpoint protein CDC25A. These results reveal hyperactive DNA damage responses upon FA loss which may underlie the attenuated cell cycle progression of FA-iPSC independent of p53. Remarkably, effects in this FA model system appear equivalent to those responsible for the depletion of HSC in the bone marrow of FA patients. Thus, iPSC models may be useful for future studies of the mechanisms underlying FA stem cell arrest and for the development of therapeutics that alleviate these phenotypes. DisclosuresNo relevant conflicts of interest to declare.

IL-6, IL-8, MMP-2, MMP-9 are overexpressed in Fanconi anemia cells through a NF-κB/TNF-α dependent mechanism.

Fanconi anemia (FA) is a rare autosomal recessive genetic disorder associated with a bone-marrow failure, genome instability, hypersensitivity to DNA crosslinking agents and a predisposition to cancer. Mutations have been documented in 16 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its symptoms. FA cells have been characterized by an overproduction of cytokines, MAPKs, and Interleukins. Through this study we have identified the overexpression of additional secretory factors such as IL-6, IL-8, MMP-2, and MMP-9 in FA cells and in cells depleted of FANCA or FANCC and proved that their expression is under the control of NF-κB/TNF-α signaling pathways. We also demonstrated that these overexpressed secretory factors were effective in promoting the proliferation, migration, and invasion of surrounding tumor cells a fundamental event in the process of epithelial mesenchymal transition (EMT) and that they also modulated the expression of EMT markers such as E-cadherin and SNAIL. Overall our data suggest that the upregulation of EMT promoting factors in FA may contribute to predisposing FA patients to cancer, thereby providing new insights into possible therapeutic interventions.

High-risk human papillomavirus E6 protein promotes reprogramming of Fanconi anemia patient cells through repression of p53 but does not allow for sustained growth of induced pluripotent stem cells.

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway, which promotes error-free repair of DNA double-strand breaks, is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus, cells from Fanconi anemia patients, which lack this critical pathway, fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6, but not E7, recovers FA iPSC colony formation and, furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.

Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

FANCD2 and CtIP Cooperate to Repair DNA Interstrand Crosslinks

The resolution of DNA interstrand crosslinks (ICLs) requires a complex interplay between several processes of DNA metabolism, including the Fanconi anemia (FA) pathway and homologous recombination (HR). FANCD2 monoubiquitination and CtIP-dependent DNA-end resection represent key events in FA and HR activation, respectively, but very little is known about their functional relationship. Here, we show that CtIP physically interacts with both FANCD2 and ubiquitin and that monoubiquitinated FANCD2 tethers CtIP to damaged chromatin, which helps channel DNA double-strand breaks generated during ICL processing into the HR pathway. Consequently, CtIP mutants defective in FANCD2 binding fail to associate with damaged chromatin, which leads to increased levels of nonhomologous end-joining activity and ICL hypersensitivity. Interestingly, we also observe that CtIP depletion aggravates the genomic instability in FANCD2-deficient cells. Thus, our data indicate that FANCD2 primes CtIP-dependent resection during HR after ICL induction but that CtIP helps prevent illegitimate recombination in FA cells.

Primary intracranial leiomyosarcoma of the torcular Herophili associated with Fanconi anemia and allogenic stem cell transplantation

Fanconi anemia is associated with a high risk for developing malignant tumors. The occurrence of primary intracranial leiomyosarcoma, however, which in general has a poor prognosis, has not been described thus far. The purpose of this study is to report on management and outcome of leiomyosarcoma of the torcular Herophili associated with Fanconi anemia in a pediatric patient. A 12-year-old girl with Fanconi anemia presented with a primary intracranial leiomyosarcoma arising from the torcular Herophili and infiltrating the adjacent venous sinuses after previous allogenic hematopoietic stem cell transplantation. Radical tumor resection followed by radiotherapy resulted in tumor-free survival and good outcome at a 2-year follow-up. Despite occurrence of leiomyosarcoma in a site thought unfavorable for surgery, combined tumor resection and radiosurgery may yield excellent outcome.

Radiation-induced mitotic catastrophe in FANCD2 primary fibroblasts

Purpose: As the Fanconi anemia (FA) pathway is required for appropriate cell cycle progression through mitosis and the completion of cell division, the aim of the present study was to determine the destiny of FA cells after irradiation in vitro and to elucidate any difference in radiosensitivity between FA and control cells.Materials and methods: Analyses of phosphorylated histone H2AX (γ-H2AX) foci, micronuclei formation and cell cycle analysis were performed in unirradiated (0 min) and irradiated primary FA fibroblasts and in a control group at different post-irradiation times (30 min, 2 h, 5 h and 24 h).Results: The accumulation of γ-H2AX foci in irradiated FA fibroblasts was observed. At 24 h post-irradiation, 57% of FA cells were γ-H2AX foci-positive, significantly higher than in the control (p < 0.01). The cell cycle analysis has shown the transient G2/M arrest in irradiated FA fibroblasts. The portion of cells in the G2/M phase showed initial increase at 30 min post-irradiation and afterwards decreased over time reaching the pretreatment level 24 h after irradiation. Irradiated FA fibroblasts progressed to abnormal mitosis, as is shown by the production of cells with different nuclear morphologies from binucleated to multinucleated surrounded with micronuclei, and also by a high percentage of foci-positive micronuclei. The majority of radiation-induced micronuclei were γ-H2AX foci-positive, indicating that radiation-induced micronuclei contain fragments of damaged chromosomes. In contrast, in the control group, most of the micronuclei were classified as γ-H2AX foci-negative, which indicates that cells with unrepaired damage were blocked before entering mitosis.Conclusion: The results clearly indicate that mitotic catastrophe might be an important cell-death mechanism involved in the response of FA fibroblasts to ionizing radiation.

Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells

FANCD2 is required for the repair of DNA damage by the FA (Fanconi anemia) pathway, and, consequently, FANCD2-deficient cells are sensitive to compounds such as cisplatin and formaldehyde that induce DNA:DNA and DNA:protein crosslinks, respectively. The DNA2 helicase/nuclease is required for RNA/DNA flap removal from Okazaki fragments during DNA replication and for the resection of DSBs (double-strand breaks) during HDR (homology-directed repair) of replication stress-induced damage. A knockdown of DNA2 renders normal cells as sensitive to cisplatin (in the absence of EXO1) and to formaldehyde (even in the presence of EXO1) as FANCD2−/− cells. Surprisingly, however, the depletion of DNA2 in FANCD2-deficient cells rescues the sensitivity of FANCD2−/− cells to cisplatin and formaldehyde. We previously showed that the resection activity of DNA2 acts downstream of FANCD2 to insure HDR of the DSBs arising when replication forks encounter ICL (interstrand crosslink) damage. The suppression of FANCD2−/− by DNA2 knockdowns suggests that DNA2 and FANCD2 also have antagonistic roles: in the absence of FANCD2, DNA2 somehow corrupts repair. To demonstrate that DNA2 is deleterious to crosslink repair, we used psoralen-induced ICL damage to trigger the repair of a site-specific crosslink in a GFP reporter and observed that “over-resection” can account for reduced repair. Our work demonstrates that excessive resection can lead to genome instability and shows that strict regulatory processes have evolved to inhibit resection nucleases. The suppression of FANCD2−/− phenotypes by DNA2 depletion may have implications for FA therapies and for the use of ICL-inducing agents in chemotherapy.

Prevalence of FA-D2 Rare Complementation Group of Fanconi Anemia in Serbia

To investigate genetic subtypes of inherited bone marrow failure syndrome Fanconi anemia (FA) in Sebia. FA-D2 subtype was found to be the most frequent genetic subtype among investigated FA patients; specific observations of FA-D2 phenotype are pointed out. Several biological endpoints of FA cells in vitro such as radiation-induced level of lymphocyte micronuclei (radiosensitivity), base line and radiation induced level of the DNA double strand breaks (DSBs), leukocyte apoptosis, and telomere capping function were assessed. The results indicate that all FA-D2 patients display radioresistant in vitro response, which is seen as significantly reduced yield of radiation-induced micronuclei. On the contrary, FA-A patients display radiosensitive in vitro response seen as increased number of radiation-induced micronuclei (MN). A massive elimination of irradiated cells via apoptosis is found in both FA-A and FA-D2 subtypes. In FA-A subtype apoptosis positively relates with the yield of radiation-induced MN, whereas in FA-D2 subtype apoptosis relates with a high percentage of cells carrying dysfunctional telomeres. The present results unequivocally demonstrate that cytokinesis-block micronucleus (CBMN) assay and analyses of telomere capping function can be used to distinguish FA-D2 and FA-A complementation groups. Considering all biological endpoints were analyzed, it can be concluded that all FA patients are radiosensitive, regardless of their complementation group. Thus, using CBMN test and telomere capping function analysis can discriminate FA-A from FA-D2 complementation groups, which could be important for assessment the conditioning regimens prior to bone marrow transplantation.

Epigenetic Alterations In Fanconi Anemia: Role In Disease Progression and Therapeutic Potential

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability, progressive bone marrow failure and increased incidence of haematological and non-haematological malignancies. Mutations in 16 FA genes have been identified which disrupt a DNA repair complex, resulting in increased chromosomal fragility. However, the phenotype is variable even amongst patients from the same family and with the same mutation. This raises the possibility that other factors, such as epigenetic modifications, may affect the disease phenotype.Epigenetic changes play an important role in oncogenesis of several haematological malignancies and solid tumours. These changes may be pharmacologically manipulated with DNA hypomethylating agents and histone deacetylase inhibitors.The aim of our project was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype.To assess whether epigenetic patterns in FA patients differ from unaffected individuals, we quantified the expression levels of genes involved in epigenetic modifications in peripheral blood mononuclear cells (PBMNC) from 12 FA patients and 15 unaffected individuals using a 84 epigenetic gene PCR array. Compared to unaffected cells, FA patients have decreased expression levels of genes involved in DNA methylation (DNMT1, DNM3Tβ), genes encoding proteins that regulate the activity of histones by acetylation (CIITA), phosphorylation (PAK1), ubiquitination (RNF20), deacetylation (HDAC2, HDCA8, HDAC9, HDAC10, HDAC11) and methylation (SETD6).We also assessed methylation status of tumor suppressor gene promoters in one FA patient and found that the FA patient had global hypomethylation in the promoter regions of tumour suppressor genes as compared to non-FA individuals, which is an early frequent event in cancer, and correlates with severity of oncologic disease.The same epigenetic gene PCR array was used to study of the effect of Vorinostat in the expression of these genes in FA-PBMNC. Vorinostat significantly increased the expression of DNM3Tβ in these cells. Vorinostat reduced expression of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation.Given the ability of Vorinostat to modulate epigenetic regulator genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by quantifying FA cell hypersensitivity towards DNA cross-linking agents following incubation with Vorinostat. FA-PBMNC pre-treated with Vorinostat showed a significant reduction of the frequency of cells with breaks induced by DEB (25,7% ± 3,5%) compared to DEB treatment alone (42,8% ± 6,5%).The effect of Vorinostat in cell cycle progression and apoptosis was evaluated by flow cytometry in FA lymphoblastoid cell lines. Vorinostat increases the number of cells in the G1 phase decreasing those in the S phase and induces apoptosis. Treatment of these cells with N-acetylcysteine protects from Vorinostat-induced apoptosis but not cell cycle arrest, suggesting reactive oxygen species may play a role in these processes.Our results identify different epigenetic patterns in FA cells relative to non-FA cells, suggesting that epigenetic changes might play a role in the pathophysiology of FA. The reduction in chromosomal breaks by Vorinostat suggests this therapeutic strategy may be useful in FA. Disclosures:No relevant conflicts of interest to declare.